Bovine viral diarrhea (BVD) is one of the main multi syndrome diseases affecting cattle that results in a significant economic impact to the cattle industry. Outbreaks of BVD are frequent and global. Economic loss in beef production in affected farms can be in the range of $31-$60 per animal depending on the type of animal production. Many countries in the European Union and businesses in the United States have adopted control programs which are mainly based on vaccination and testing/removing carrier animals.
Common manifestations of BVD include: abortions, infertility, irregular heat cycles, early embryonic deaths, fetal mummification, immunosuppression, diarrhea, fever, pneumonia, and other potentially fatal conditions. BVD cannot be clinically distinguished from other diseases which are manifested with similar symptoms. Immunosuppression lowers the resistance of infected animals to other common pathogens which leads to numerous indirectly caused clinical manifestations in cattle, most commonly bovine respiratory syndrome (BRS). Direct and mostly indirect clinical effect on cattle herds lead to significant economic loss. In rare cases animals can be acutely infected with severe manifestations of the disease. Also animals can be persistently infected when they serve as the reservoir of the infection for other animals.
An animal becomes persistently infected (PI) with Bovine Viral Diarrhea Virus (BVDV) if the fetus is exposed to slow-growing, low-virulence strains of the virus between days 30 and 125 of gestation. Fetuses exposed to BVDV after 125 days of gestation will mount an immune response against the virus, which clears the infection and usually develop quite normally. Fetuses exposed to a rapidly growing or “HOT” strain of the virus are usually killed. PI animals lack immunity to BVDV and are lifetime carriers of the virus. PI animals shed several billion viral particles a day and serve as a reservoir of BVDV in a herd. Animals that are exposed to BVDV acutely may become infected and shed a virus for a few days until they present an immune response. These animals recover from the infection and do not remain carriers.
Strategies for control of BVDV include vaccination, management practices, and most importantly strict biosafety measures. Vaccination is not very effective due to the high variability of the virus which causes BVD. Biosafety measures involve testing of all animals which are introduced to the herd and separation of all PI animals so that they cannot cause infection in naive cattle (non-infected). Non-specific symptoms and failure of field vaccinations for BVDV increase the need for a test protocol that will help identify and eliminate carrier PI animals in a cost-effective manner.
BVD is caused by BVDV. BVDV is an umbrella term for a diverse group of viruses in the genus pestivirus of the family Flaviviridae. It is further classified into two different genotypes known as BVDV1 and BVDV2, which represent two distinct species. Within each genotype there are many different strains of the virus that differ significantly in their pathogenesis. Severe acute disease has only been reported with small number of BVDV2 strains. In addition, BVDV is classified into three different biotypes based on their cytopathic effect when grown in vitro: cytopathic which degenerate epithelial cells in vitro, non-cytopathic which does not degenerate epithelial cells in vitro and lymphocytopathic which degenerates lymphocytes in vitro, but it does not degenerate epithelial cells (Ridpath et al., Lymphocytopathogenic activity in vitro correlates with high virulence in vivo for BVDV type 2 strains: Criteria for a third biotype of BVDV. Virus Res. 2006). Lymphocytopathic biotype correlates with high virulence in acute infections. Due to the aforementioned factors BVDV is not to be limited to a specific strain of virus, but rather refers to an umbrella of pathogenic and benign organisms within the genus Pestivirus.
Organisms in the genus Pestiviruses have a positive sense single stranded RNA genome (SS+RNA). Organisms referred to as BVDV contain a genome of approximately 12,500 nucleotides with a 5′-nontranslated region (NTR), a single large open reading frame (ORF), and a 3′-NTR lacking a poly(A)tail. The 5′-NTR contains an internal ribosome entry site that initiates translation of BVDV mRNA. The secondary structure of the 5′-NTR is involved in the regulation of translation and genome replication. The genomic RNA has one open reading frame (ORF) of about 4000 codons whose translation yields one precursor poly protein, which is co- and post translationally cleaved into 11 or 12 mature proteins, by viral and host cell encoded proteases (“processing”). Most of the virally encoded cleaving is catalyzed by a serine protease domain within the non-structural protein NS3 and generates the non-structural proteins NS3 to NS5B, whereas the structural proteins are believed to be cleaved by cellular proteases.
BVDV virions consist of four structural proteins (Meyers et al., Molecular characterization of pestiviruses. Adv Virus Res. 1996); nucleocapsid C protein, envelope glycoproteins Erns, E1 and E2. Along with the structural proteins described above, the viral genome encodes several non-structural proteins (Npro, p7, NS2/3, (NS2, NS3), NS4A, NS4B, NS5A and NS5B) which are essential for replication of the virus. BVDV proteins and their function are described in TABLE 1.
TABLE 1CCapsid protein (core protein).ErnsEnvelope glycoprotein (rns means Rnasesecreted); induces production of antibodieswith a weakneutralizing activity.E1Envelope glycoprotein.E2Envelope glycoprotein; it features epitopesthat are recognized by the host immune system.Antibodies against these epitopes areessential for the neutralization of viralinfectivity.p7Very small protein with largely unknownfunction. Essential for the formation ofinfective virus particles.NproThe N-terminal protein of BVDV codes for acysteine protease that cleaves the N-terminusfrom the core protein (auto-protease).NS2/3Serine protease; biggest BVDV-protein with amolecular weight of 125 kD; cytopathic BVDVdoes not only express NS2/3 in vitro in onepiece but also in two separate proteins (NS2,54 kD and NS3, 80 kD, commonly called p80). Inthis application and the appended claims,NS2/3 will be used to reference NS2/3, NS2,NS3 or any part of these molecules, unlessotherwise stated.NS4A/BNS4A is a cofactor for serine protease NS2/3;there's evidence that NS4B plays a role inviral cytopathogenicity; both do not induce animmune response.NS5ANS5A is part of the replication complex.NS5BNS5B is RNA dependent RNA-polymerase.
Diagnostic tests for BVD are oriented toward detection of the presence of viral antigens or viral RNA in bodily fluids or tissue samples. Positive animals are considered carriers of BVDV and are separated from other animals to prevent disease spread through the herd. One sole PI animal has the ability to infect every animal in direct contact. Given this information it is essential that diagnostic tests have very high sensitivity in order to adequately identify every PI animal.
Current BVDV detection methods include: Polymerase Chain Reaction (PCR), standard viral isolation techniques (i.e. culture), immunohistochemistry, and antigen capture enzyme-linked immunoassay (ELISA). PCR technology amplifies and detects the viral RNA. This makes PCR extremely sensitive, but also this method is prohibitively expensive to the cattle industry. In order to counter the expense the laboratories have decided to batch several individual samples together to create a pooled sample, and then test the pooled sample. The inherent problem with this approach is that if the pool sample is positive for the virus the lab cannot determine which individual samples within the pooled sample contain the virus. In this instance all of the original samples must be retested individually to determine which cattle are affected. Also inhibitory factors can lead to false negative PCR reactions. This problem is amplified by the use of pooled samples. Viral RNA is relatively unstable and false negative reactions can be observed. All those limitations lead to lower sensitivity and questionable usefulness of pooled PCR technique (Edmondson et al., Comparison of tests for detection of bovine viral diarrhea virus in diagnostic samples. J Vet Diagn Invest. 2007). Viral isolation by culture is costly, very complex, time consuming, and can only be performed in a few specialized laboratories limiting its appeal. Immunohistochemistry is also a costly, very complex, and labor intensive technique reserved only for specialized laboratories also limiting its appeal.
ELISA technology however is well suited as a broad based diagnostic tool because it is relatively inexpensive, simple, reproducible and can be performed almost anywhere. Currently ELISA testing for the presence of the viral antigens is done in most cases using ELISA system that detects Erns protein. Erns represents a structural glycoprotein and as such is a part of the envelope of the virus. Thus, it is present in infected cells, as well as outside of infected cells in circulation. Sensitivity of this ELISA system based on the detection of Erns protein is in the range of 99.7% per our experiments on animals three months or older. This technology is patented (U.S. Pat. No. 7,449,288) and is available from one commercial supplier. There are also a minimum of three ELISA tests available, based on the detection of NS2/3 protein. Commercially those tests are not significant because they generally show lower sensitivity then Erns based ELISA (95.5% sensitivity according to our experiments and also according to some external studies (IDEXX Laboratories, internal data). Recently published papers show that NS2/3 protein and its derivative NS3 protein are not suitable for detecting BVDV in cattle because: NS3 molecule is unstable, it is not uniformly distributed in tissues and there is only a small quantity of it to detect (R. A. Fux, Dissertation, University of Munich, 2007). The disclosed methodology presents a much improved method of detecting non-structural proteins such as NS2/3. Disclosed technology overcomes problems of solubility of NS2/3 protein, its stability and drastically increases the quantity of the available antigen for detection with NS2/3 specific antibodies.